This invention is in the field of mass spectrometry, more particularly to methods of data acquisition in tandem mass spectrometry.
The ability to identify proteins and determine their chemical structures has become central to the life sciences. The amino acid sequence of proteins provides a link between proteins and their coding genes via the genetic code, and, in principle, a link between cell physiology and genetics. The identification of proteins provides a window into complex cellular regulatory networks.
Mass spectrometry (MS) is commonly used to provide information related to protein composition and peptide sequence. As efforts shift from sequencing the genome to understanding and identifying expressed genes and protein function, it is increasingly important that analytical tools be developed for providing reliable and rapid protein sequencing. Such protein sequence information can be used in proteomic databases and for identifying, understanding and using sequence information in a wide range of applications from fundamental research to medical treatment.
Ion trap mass spectrometers are among the most widely used platforms for molecular analysis—spanning natural products, to pharmaceuticals, to biologics such as proteins. Most mass spectrometer-based experiments begin with the isolation of a group of compounds from a set of samples through some sort of extraction technique, e.g., proteins from tissues, cell lysates, or fluids followed by proteolytic digestion of those proteins into peptides. Frequently, but not necessarily, the mass spectrometers are coupled with some form of separations, e.g., electrophoretic or chromatographic. Over the course of just a few hours, mass spectral instruments can autonomously interrogate tens of thousands of molecular species.
In tandem mass spectrometry (MS/MS or MS2), multiple rounds of mass spectrometry analysis are performed. For example, samples containing a mixture of proteins and peptides can be ionized and the resulting precursor ions separated according to their mass-to-charge ratio. Selected precursor ions can then be fragmented and further analyzed according to the mass-to-charge ratio of the fragments.
Technical developments in chromatography and MS instrumentation have made two types or protein sequencing methods popular: (1) the bottom-up approach and (2) the top-down approach. For the bottom-up approach, a protein-containing sample is digested with a proteolytic enzyme resulting in a complex mixture of peptides. Next, the digested sample is chromatographically separated (in one or multiple dimensions) and introduced to an electrospray ionization (ESI) source on the mass spectrometer. The ESI source converts condensed phase ions, eluting from the HPLC column, to multiply-protonated molecules (cations) in the gas-phase—a requirement for MS analysis. The mass spectrometer first records the mass/charge (m/z) of each peptide ion and then selects the peptide ions individually to obtain sequence information via MS/MS. In a typical shotgun proteomics experiment a cell lysate, containing as many as several thousand proteins, is analyzed. In the top-down method intact proteins are ionized and directly sampled by the mass spectrometer and then fragmented during MS/MS analysis.
Liquid chromatography coupled to MS/MS is arguably the most common and most effective method for global identification of peptides and proteins. The m/z peaks corresponding to the precursor ions are plotted with respect to intensity on a mass spectrum, and represent fragments that can be further analyzed to identify peptides and proteins of interest. For complex mixtures, however, not all precursor ions can be selected for further analysis within a given elution window. Furthermore, many of the peaks and corresponding precursor ions do not lead to successful identification of the protein or peptide. The most common solution to this has been to select peaks in order of decreasing intensity. The precursor ions having the least intense peaks are then excluded from consideration for a set amount of time.
However, selecting precursor ions solely by intensity of their initial MS peaks does not always result in the selection of precursor ions most likely to lead to successful identification of the protein or peptide. Selecting precursor ions using another characteristic, such as their mass-to-charge ratio, may be more advantageous depending on the sample and MS/MS conditions, but similarly may not result in the selection of precursor ions most likely to lead to successful identification.
Moreover, selection of the precursor ions may occur when the attributes of some precursor ions are not ideal (e.g., they exhibit low intensity or are observed in the presence of other more abundant precursors with similar m/z ratios) thus precluding their identification. Almost all modern LC-MS/MS methods employ some version of “dynamic exclusion”. Dynamic exclusion ensures that once a precursor ion is selected for fragmentation and subsequent MS2 analysis, it is excluded from further MS2 selection for a fixed, user-defined amount of time (typically 30-90 seconds). The rationale is that there are many precursor ions to interrogate and it is inefficient to repeat MS2 analysis on the same precursor ions multiple times. However, these methods are based on some basic assumptions that are very often not true: (1) that the likelihood of a successful identification is constant for the entire period of time that the precursor ion is observed in MS1 scans; and (2) that there are always new precursor ions to select for fragmentation and MS2 analysis.
In many cases, a precursor ion can be selected for MS2 analysis when its MS1 intensity is very low or when it could be observed in the presence of a much more abundant precursor a mere 0.3 Daltons away. The probability that the precursor ion will be successfully identified from the MS2 scan during this time period may be very low. However, twenty seconds later it might be much more intense and the neighboring precursor ion might not be present any more. In such cases, it may be worth performing another MS2 analysis on this precursor ion. This is especially true if the duty cycle on the mass spectrometer is extremely fast and has already selected all of the peaks in the MS1 window for MS2 analysis, which can be observed with modern mass spectrometers. By excluding these precursor ions from consideration via dynamic exclusion there is no opportunity to reselect them for MS2 analysis when their attributes are more suited for identification.
What is needed is an improved method of data acquisition that enables the selection of precursor ions more likely to lead to successful identification, including precursor ions which may normally be excluded from further analysis.